The work leading to the present invention was partially supported by National Heart, Lung and Blood Institute Grants Nos. HL28972 and HL41256, and Contract No. HV53030. The government may hold rights in the present patent.
1. Field of the Invention
This invention relates to an endogenous baboon plasma cholesteryl esters transfer protein (CETP) inhibitor polypeptide. More specifically, this invention relates to the identification and characterization of the polypeptide and to novel synthetic peptides possessing inhibitory activity of CETP. The endogenous inhibitory peptide has a molecular weight of 4000, is present in plasma in the form of modified apo A-I and apo E having molecular weights of 31kD and 41kD, respectively, and has a common amino acid sequence with the N-terminal fragment of apo C-I. This invention also relates to an anti-atherosclerosis composition, a kit, and to antibodies raised against the N-terminal amino acid sequence of the inhibitory polypeptide. The inhibitory peptide of the invention, fragments thereof and analogues thereof are useful for the prophylactic and therapeutic treatment of atherosclerosis.
2. Description of the Background
Atherosclerosis is one of the most widespread health problems in the United States today as are its attendant complications, particularly coronary heart disease. A number of risk factors have been associated with the development of premature atherosclerosis, primarily elevated plasma cholesterol levels. Due to the crucial role cholesterol appears to play in the occurrence of heart disease, a great deal of attention has been devoted to studying its synthesis, transport and metabolism in the human body.
Of particular interest is the establishment of relationships between the levels of plasma lipoproteins or serum lipids and the risk of development of coronary heart disease. Both high density lipoproteins (HDL) and low density lipoproteins (LDL) carry cholesterol mainly in the form of cholesteryl esters (CE). There are some indications, however, that while LDL cholesterol is a positive risk factor, HDL cholesterol is an even more important negative risk factor. Although the exact functions of these lipoproteins have not been completely established, HDL appears to serve for the removal of cholesterol from peripheral cells and its transport back to the liver, where a large proportion of the cholesterol excreted from the body is removed.
LDL and HDL are believed to play key roles in the development of cardiovascular disease by overloading the lysosomes of the walls of arterial cells with metabolites which are generally hydrolyzed slowly, such as CE and triglycerides. These products are evacuated from the liver and intestine by plasma LDL. When the amount of lipids to be transported exceeds the transporting capacity of HDL to the liver for excretion, CE become deposited in the cells in certain critical areas, such as arterial walls. This overloading eventually results in impaired cell function, and if continued may produce cell death. A continuous overloading results in the accumulation of cellular debris and tile formation of atherosclerotic plaque in the vessel wall. This, in turn, leads to the blockage of the affected artery and/or muscular spasm, events which may manifest themselves as coronary heart disease or strokes. Thus, the level of HDL in plasma has been negatively correlated with the probability of developing atherosclerosis in humans and experimental animals.
Although the level of HDL has been shown to vary considerably among individuals, the means of regulation of such plasma level remains to be elucidated.
CETP transfers CE from HDL to VLDL and LDL, and it has been suggested that it plays an important role in the regulation of plasma HDL levels. Some hyperalphalipoproteinemic patients were reported to have high levels of large HDL particles that were clearly separate from LDL. Plasma samples from these patients were shown to lack CETP activity (Koizumi et al, Atherosclerosis 58:175-186(1985)). A homozygous subject with familial hyperalphalipoproteinemia was found to have impaired transfer of CE from HDL to LDL (Yokoyama et al., Artery 14:43-51(1986)). A fraction of density d&gt;1.21 g/ml from the subject's plasma evidenced substantial CETP activity with normal HDL. The HDL, however, proved to be a poor substrate for CETP.
Certain animal sires and their progeny possess unusual lipoproteins patterns, e.g., lipoproteins of a density intermediate to that of LDL and HDL, or large high density lipoproteins. These lipoproteins have been designated HDL.sub.1, and the animal phenotype as "high HDL.sub.1 ". Baboon strains possessing, for instance, patterns of either high or low HDL.sub.1 are known. In most cases, HDL.sub.1 separates either as a distinct peak between LDL and HDL or as a shoulder to the HDL peak, and is induced by a high cholesterol, high lard (HCHF) diet. The proportion of HDL.sub.1 diminishes when the baboons are fed a diet that is either enriched ill polyunsaturated fat, with or without cholesterol. Occasionally, however, the amount of HDL.sub.1 present in high HDL.sub.1 baboons fed the chow diet is low.
In some baboon families, the level of plasma HDL.sub.1 was shown to increase when the animals are challenged with a HCHF diet. When fed a HCHF diet, the baboons also show higher plasma HDL. More generally, the accumulation of HDL in baboons as well as in humans is associated with a slower transfer of CE from HDL to very low density lipoproteins (VLDL) and LDL. Thus, baboons with high HDL.sub.1 plasma levels are excellent as animal models for the study of hyperalphalipoproteinemia.
In a previous study, some of the present inventors reported that a slower transfer of CE from HDL to VLDL and LDL was observed in high HDL.sub.1 baboons. This was attributed to the presence of a CETP protein inhibitor associated with HDL and intermediate density lipoprotein (IDL) particles (Kushwaha et al, J. L. Lipid Res. 31:965-974(1990)). An accumulation of HDL.sub.1 in the high HDL.sub.1 baboons fed a HCHF diet was reported along with a slower transfer of CE from HDL to LDL. A similar protein was found in human plasma by Son and Zilversmit (Son and Zilversmit, B.B.A. 795:473-480(1984)). The human protein has a molecular weight of 31,000 and suppresses the transfer of triacylglycerol and CE.
Several other species including rat, pig and dog, have been reported to readily accumulate HDL.sub.1 in plasma. Kurasawa et al (1985), supra, reported that a homozygous subject with familial hyperalphalipoproteinemia, has impaired CE transfer between HDL and LDL (Kurasawa et al, J. B. Biochem. 98:1499-1508(1985)). Separately, Yokoyama et al reported that a plasma fraction of d&gt;1.21 g/ml of the same subject evidenced substantial CE transfer activity when tested with normal HDL (Yokoyama et al, Artery 14(1):43-51(1986)). The HDL particles accumulated by this subject were substantially larger in molecular size than ordinary HDL.sub.2.
HDL is generally divided into subfractions based on their particle sizes and densities. These fractions include HDL.sub.1, HDL.sub.2 and HDL.sub.3. HDL.sub.1 has the largest particles and is usually not present in the plasma of normal humans or non-human primates. HDL.sub.2 and HDL.sub.3 are the normal components of human plasma. HDL.sub.2 is larger than HDL.sub.3 and differs between men and women.
Many attempts have been made to interfere with the transport and transfer of cholesterol in mammalians in order to alter its plasma levels. Among them are the following.
U.S. Pat. No. 4,987,151 to Taboc discloses triterpene derivatives that inhibit acyl coenzyme A:cholesterol acyltransferase (ACAT) enzyme. The ACAT is a cellular enzyme that is not present in plasma, and esterfies cellular cholesterol to form CE. This enzyme is different from the CE transfer protein (CETP) present in plasma. The CETP does not form CE as does the ACAT enzyme. Instead, the CETP transfers CE amongst different plasma lipoproteins.
U.S. Pat. No. 4,643,988 to Segrest et al discloses amphipathic peptides which are capable of substituting for apo A-I in HDL. Apo A-I is known to stimulate the lecithin cholesterol:acyl transferase (LCAT) enzyme, a plasma enzyme that forms CE in HDL. Plasma CETP, in contradistinction, transfers CE from HDL to VLDL and LDL. The function of the CETP enzyme is, therefore, different from that of the LCAT enzyme, as well. The amino acid sequences of the Segrest et al peptides are, in addition, different from the sequences of the CETP inhibitor of this invention.